Fusion proteins of Mycobacterium tuberculosis

ABSTRACT

The present invention relates to compositions and fusion proteins containing at least two Mycobacterium sp. antigens, and nucleic acids encoding such compositions and fusion proteins. The compositions of the invention increase serological sensitivity of sera from individuals infected with tuberculosis, and methods for their use in the diagnosis, treatment, and prevention of tuberculosis infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. S No. 60/357,351,filed Feb. 15, 2002, herein incorporated by reference in its entirety.

[0002] The present application incorporates by reference the followingapplications in their entirety: U.S. patent application Ser. No.09/056,556, filed Apr. 7, 1998; U.S. patent application Ser. No.09/223,040, filed Dec. 30, 1998; U.S. patent application Ser. No.09/287,849, filed Apr. 7, 1999; published PCT application No.WO99/51748, filed Apr. 7, 1999 (PCT/US99/07717); U.S. patent applicationNo. 60/158,338, filed Oct. 7, 1999; U.S. patent application No.60/158,425, filed Oct. 7, 1999; U.S. patent application Ser. No.09/597,796, filed Jun. 20, 2000; U.S. patent application Ser. No.09/688,672, filed Oct. 10, 2000; published PCT application No. WO01/24820, filed Oct. 10, 2000 (PCT/US00/28095); U.S. patent applicationNo. 60/265,737, filed Feb. 1, 2001; U.S. patent application Ser. No.09/886,349, filed Jun. 20, 2001; and published PCT application No.WO01/98460, filed Jun. 20, 2001 (PCT/US01/19959).

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] The present invention relates to fusion proteins containing atleast two Mycobacterium sp. antigens. In particular, it relates tonucleic acids encoding fusion proteins that include two or moreindividual M. tuberculosis antigens, which increase serologicalsensitivity of sera from individuals infected with tuberculosis, andmethods for their use in the diagnosis, treatment, and prevention oftuberculosis infection.

BACKGROUND OF THE INVENTION

[0005] Tuberculosis is a chronic infectious disease caused by infectionwith M. tuberculosis and other Mycobacterium species. It is a majordisease in developing countries, as well as an increasing problem indeveloped areas of the world, with about 8 million new cases and 3million deaths each year. Although the infection may be asymptomatic fora considerable period of time, the disease is most commonly manifestedas an acute inflammation of the lungs, resulting in fever and anonproductive cough. If untreated, serious complications and deathtypically result.

[0006] Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance.

[0007] In order to control the spread of tuberculosis, effectivevaccination and accurate early diagnosis of the disease are of utmostimportance. Currently, vaccination with live bacteria is the mostefficient method for inducing protective immunity. The most commonmycobacterium employed for this purpose is Bacillus Calmette-Guerin(BCG), an avirulent strain of M. bovis. However, the safety and efficacyof BCG is a source of controversy and some countries, such as the UnitedStates, do not vaccinate the general public with this agent.

[0008] Diagnosis of tuberculosis is commonly achieved using a skin test,which involves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48-72 hours after injection, whichindicates exposure to mycobacterial antigens. Sensitivity andspecificity have, however, been a problem with this test, andindividuals vaccinated with BCG cannot be distinguished from infectedindividuals.

[0009] While macrophages have been shown to act as the principaleffectors of Mycobacterium immunity, T cells are the predominantinducers of such immunity. The essential role of T cells in protectionagainst Mycobacterium infection is illustrated by the frequentoccurrence of Mycobacterium infection in AIDS patients, due to thedepletion of CD4⁺ T cells associated with human immunodeficiency virus(HIV) infection. Mycobacterium-reactive CD4⁺ T cells have been shown tobe potent producers of γ-interferon (IFN-γ), which, in turn, has beenshown to trigger the anti-mycobacterial effects of macrophages in mice.While the role of IFN-γ in humans is less clear, studies have shown that1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ ortumor necrosis factor-alpha, activates human macrophages to inhibit M.tuberculosis infection. Furthermore, it is known that IFN-γ stimulateshuman macrophages to make 1,25-dihydroxy-vitamin D3. Similarly,interleukin-12 (IL-12) has been shown to play a role in stimulatingresistance to M. tuberculosis infection. For a review of the immunologyof M. tuberculosis infection, see Chan & Kaufmann, Tuberculosis:Pathogenesis, Protection and Control (Bloom ed., 1994), and Harrison 'sPrinciples of Internal Medicine, volume 1, pp. 1004-1014 and 1019-1023(14th ed., Fauci et al., eds., 1998).

[0010] Accordingly, there is a need for improved diagnostic reagents,and improved methods for diagnosis, preventing and treatingtuberculosis.

SUMMARY OF THE INVENTION

[0011] The present invention comprises two novel fusion proteinscontaining at least two Mycobacterium sp. antigens. Specifically thenucleic acids encode two fusion polypeptides: MTB32Mut-39F and MTB102F.MTB32Mut-39F includes a mutated MTB32A antigen and a MTB39 antigen(TBH9). MTB102F includes a mutated MTB32A antigen, a MTB39 antigen, anda 85B antigen. The inventors of the present application surprisinglydiscovered that MTB32Mut SA-39F and MTB102F are expressed at higherlevels, are more stable, and are more immunogenic than other M.tuberculosis antigens.

[0012] One embodiment of the present invention is an isolated nucleicacid encoding a fusion polypeptide comprising a MTB32Mut antigen and aMTB39 (TBH9) antigen from a Mycobacterium species of the tuberculosiscomplex. The nucleic acid hybridizes under highly stringent conditionsto a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1 or acomplement thereof. The MTB32Mut antigen has a mutation at amino acidposition 183 as compared to wild type MTB32A. In one embodiment, themutation is a serine to alanine mutation. The nucleic acid may comprisea nucleotide sequence SEQ ID NO:1. The nucleic acid may encode an aminoacid sequence of SEQ ID NO:2. In another embodiment, the fusion proteinfurther comprises an 85B antigen from a Mycobacterium species of thetuberculosis complex. In another embodiment, the nucleic acid comprisesSEQ ID NO:3 and encodes an amino acid sequence of SEQ ID NO:4. TheMycobacterium may be Mycobacterium tuberculosis. An expression vectormay comprise the nucleic acid. A host cell may comprise the expressionvector. The host cell may be selected from the group consisting of E.coli, yeast, and mammalian cells.

[0013] Another embodiment of the present invention is an isolated fusionprotein encoded by an isolated nucleic acid encoding a fusionpolypeptide comprising a mutated MTB32A antigen and a MTB39 antigen froma Mycobacterium species of the tuberculosis complex.

[0014] Yet another embodiment of the present invention is a compositioncomprising a an isolated nucleic acid encoding a fusion polypeptidecomprising a mutated MTB32A antigen and a MTB39 antigen from aMycobacterium species of the tuberculosis complex, as described above,and a physiologically acceptable carrier. The fusion polypeptide encodedby the nucleic acid may further comprise an NS1 antigen or animmunogenic fragment thereof. The Mycobacterium species may beMycobacterium tuberculosis.

[0015] Even still another embodiment of the present invention is acomposition comprising a mutated MTB32A antigen and a MTB39 antigen froma Mycobacterium species of the tuberculosis complex, as described above,and a physiologically acceptable carrier. The composition may furthercomprise a non-specific immune response enhancer. The nonspecific immuneresponse enhancer maybe an adjuvant. The adjuvant may comprise QS21 andMPL in an oil in water emulsion, e.g., with squalene and tocopherol,optionally including CpG. The adjuvant may be selected from the groupconsisting of ENHANZYN, MPL, 3D-MPL, IFA, QS21, CpG, CWS, TDM, AGP, CPG,Leif, saponin, and saponin mimetics. The composition may furthercomprise BCG or pVac. The composition may further comprise an NS1antigen or an immunogenic fragment thereof. The Mycobacterium speciesmay be Mycobacterium tuberculosis.

[0016] Another embodiment of the present invention is a method fordetecting tuberculosis in a patient. The dermal cells of a patient arecontacted with one or more polypeptides encoded by a nucleic acidencoding a fusion polypeptide comprising a mutated MTB32A antigen and aMTB39 antigen from a Mycobacterium species of the tuberculosis complex,as described above. The immune response is detected on the patient'sskin and therefrom tuberculosis is detected in the patient. The immuneresponse may be induration.

[0017] Even another embodiment of the present invention is a diagnostickit comprising a polypeptide encoded by a nucleic acid of the inventionand an apparatus sufficient to contact the polypeptide encoded bynucleic acid with the dermal cells of a patient.

[0018] Still another embodiment of the present invention is a method foreliciting an immune response in a mammal. An immunologically effectiveamount of a nucleic acid encoding a mutated MTB32A antigen and a MTB39antigen from a Mycobacterium species of the tuberculosis complex, asdescribed above, is administered to the mammal. The mammal may have beenimmunized with BCG. The mammal may be a human. The composition may beadministered prophylactically. The nucleic acid may comprise nucleotidesequence SEQ ID NO:1. The nucleic acid may encode an amino acid sequenceof SEQ ID NO:2. In one embodiment, the nucleic acid encoding the fusionprotein is first administered, and then a fusion protein booster islater provided.

[0019] In another embodiment, the invention provides a method foreliciting an immune response in a mammal, the method comprising the stepof administering to the mammal an immunologically effective amount of acomposition comprising a mutated MTB32A antigen and a MTB39 antigen froma Mycobacterium species of the tuberculosis complex, as described above.The mammal may have been immunized with BCG. The mammal may be a human.The composition may be administered prophylactically. In one embodiment,the fusion protein is first administered, and then a nucleic acidencoding the fusion protein is later provided as a booster.

[0020] Another embodiment of the present invention is an isolatednucleic acid encoding a fusion polypeptide comprising a mutated MTB32Aantigen, a MTB39 antigen, and a 85B antigen from a Mycobacterium speciesof the tuberculosis complex wherein said nucleic acid hybridizes underhighly stringent conditions to a nucleic acid comprising a nucleotidesequence of SEQ ID NO:3 or a complement thereof, and wherein the mutatedMTB32A antigen has a mutation at amino acid position 183 as compared towild type MTB32A. In one embodiment, the mutation is a serine to alaninemutation. The nucleic acid may comprise a nucleotide sequence SEQ IDNO:3. The nucleic acid may encode an amino acid sequence of SEQ ID NO:4.The Mycobacterium may be Mycobacterium tuberculosis. An expressionvector may comprise the nucleic-acid. A host cell may comprising theexpression vector. The host cell may be selected from the groupconsisting of E. coli, yeast, and mammalian cells.

[0021] Still another embodiment of the present invention is an isolatedfusion protein encoded by an isolated nucleic acid encoding a fusionpolypeptide comprising a mutated MTB32A antigen, a MTB39 antigen, and a85B antigen from a Mycobacterium species of the tuberculosis complex, asdescribed above.

[0022] Even still another embodiment of the present invention is acomposition comprising an isolated nucleic acid encoding a fusionpolypeptide comprising a mutated MTB32A antigen, a MTB39 antigen, and a85B antigen from a Mycobacterium species of the tuberculosis complex, asdescribed above and a physiologically acceptable carrier. The fusionpolypeptide encoded by the nucleic acid may further comprises an NS1antigen or an immunogenic fragment thereof. The Mycobacterium speciesmay be Mycobacterium tuberculosis.

[0023] Even yet another embodiment of the present invention is acomposition comprising a fusion protein comprising a mutated MTB32Aantigen, a MTB39 antigen, and a 85B antigen from a Mycobacterium speciesof the tuberculosis complex, as described above, and a physiologicallyacceptable carrier. The composition may comprise a non-specific immuneresponse enhancer. The non-specific immune response enhancer may be anadjuvant. The adjuvant may comprise QS21 and MPL, and optionally CpG.The adjuvant may be selected from the group consisting of ENHANZYN, MPL,3D-MPL, IFA, QS21, CWS, TDM, AGP, CpG, Leif, saponin, and saponinmimetics. The composition may further comprise BCG or pVac. Thecomposition may further comprise an NS1 antigen or an immunogenicfragment thereof. The Mycobacterium species may be Mycobacteriumtuberculosis.

[0024] Still another embodiment of the present invention is a method fordetecting tuberculosis in a patient. The contacting dermal cells of apatient are contacted with one or more polypeptides encoded by anisolated nucleic acid encoding a fusion polypeptide comprising a mutatedMTB32A antigen, a MTB39 antigen, and a 85B antigen from a Mycobacteriumspecies of the tuberculosis complex, as described above. An immuneresponse is detected on the patient's skin and therefrom detectingtuberculosis in the patient. The immune response may be induration. TheMycobacterium species may be Mycobacterium tuberculosis.

[0025] Even still another embodiment of the present invention is adiagnostic kit comprising an isolated nucleic acid encoding a fusionpolypeptide comprising a mutated MTB32A antigen, a MTB39 antigen, and a85B antigen from a Mycobacterium species of the tuberculosis complex, asdescribed above and an apparatus sufficient to contact the polypeptideencoded by nucleic acid with the dermal cells of a patient.

[0026] Still even another embodiment of the present invention is amethod for eliciting an immune response in a mammal, the methodcomprising the step of administering to the mammal an immunologicallyeffective amount of a nucleic acid encoding a mutated MTB32A antigen, aMTB39 antigen, and a 85B antigen from a Mycobacterium species of thetuberculosis complex, as described above. The mammal may have beenimmunized with BCG. The mammal may be a human. The composition may beadministered prophylactically. The nucleic acid may comprise nucleotidesequence SEQ ID NO:3. The nucleic acid may encode an amino acid sequenceof SEQ ID NO:4.

[0027] Yet another embodiment of the present invention is a method foreliciting an immune response in a mammal. An immunologically effectiveamount of a composition comprising a mutated MTB32A antigen, a MTB39antigen, and a 85B antigen from a Mycobacterium species of thetuberculosis complex, as described above, is administered to the mammal.The nucleic acid may comprise nucleotide sequence SEQ ID NO:3. Thenucleic acid may encode an amino acid sequence of SEQ ID NO:4. Themammal may have been immunized with BCG. The mammal may be a human. Thecomposition may be administered prophylactically.

[0028] In yet another embodiment, the present invention provides nucleicacid sequences and amino acid sequences encoding the MTB72F fusionprotein further fused to the following antigens: MAPS (fusion r95F),Erd14 (fusion MTB89F), MTI (fusion MTB83F), DPV (fusion MTB81F), mTCC#2(fusion MTB114F), hTCC#1 (fusion MTB102tm2F) and 85b complex antigenfrom M. bovis (fusion MTB103F). MTB72F fusion protein is a 72 kDapolyprotein fusion construct comprising Ra12 (C-terminus of matureRa35), TbH9, and Ra35 (N-terminus of mature Ra35 (for Ra12 and Ra35sequences, see, e.g., FIG. 19).

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows the nucleotide sequence that encodes the MTB32-MTB39Ffusion protein (SEQ ID NO:1).

[0030]FIG. 2 shows the amino acid sequence of the mutated MTB32-MTB39Ffusion protein (SEQ ID NO:2).

[0031]FIG. 3 shows the nucleotide sequence that encodes the MTB-102Ffusion protein (SEQ ID NO:3).

[0032]FIG. 4 shows the amino acid sequence of the MTB 102F fusionprotein (SEQ ID NO:4).

[0033]FIG. 5 shows the nucleic acid sequence for MTB72F-MAPS (fusionr95F; SEQ ID NO:5).

[0034]FIG. 6 shows the nucleic acid sequence for MTB72F-Erd14 (fusionMTB89F; SEQ ID NO:6).

[0035]FIG. 7 shows the nucleic acid sequence for MTB72F-MTI (fusionMTB83F; SEQ ID NO:7).

[0036]FIG. 8 shows the nucleic acid sequence for MTB72F-DPV (fusionMTB81 F; SEQ ID NO:8).

[0037]FIG. 9 shows the nucleic acid sequence for MTB72F-mTCC#2 (fusionMTB114F; SEQ ID NO:9).

[0038]FIG. 10 shows-the nucleic acid sequence for MTB72F-hTCC#1 (fusionMTB102tm2F; SEQ ID NO:10).

[0039]FIG. 11 shows the nucleic acid sequence for MTB72F and 85b complexantigen from M. bovis (fusion MTB103F; SEQ ID NO:11).

[0040]FIG. 12 shows the amino acid sequence for MTB72F-MAPS (fusionr95F; SEQ ID NO:12).

[0041]FIG. 13 shows the amino acid sequence for MTB72F-Erd14 (fusionMTB89F; SEQ ID NO:13).

[0042]FIG. 14 shows the amino acid sequence for MTB72F-MTI (fusionMTB83F; SEQ ID NO:14).

[0043]FIG. 15 shows the amino acid sequence for MTB72F-DPV (fusionMTB81F; SEQ ID NO:15).

[0044]FIG. 16 shows the amino acid sequence for MTB72F-mTCC#2 (fusionMTB114F; SEQ ID NO: 16).

[0045]FIG. 17 shows the amino acid sequence for MTB72F-hTCC#1 (fusionMTB102tm2F; SEQ ID NO:17).

[0046]FIG. 18 shows the amino acid sequence for MTB72F and 85b complexantigen from M. bovis (fusion MTB103F; SEQ ID NO:18).

[0047]FIG. 19 shows an alignment of MTB32AMutSA and wild-type MBT32A.

[0048]FIG. 20 shows an alignment of MTB72FMutSA with MTB72F.

DETAILED DESCRIPTION OF THE INVENTION

[0049] Definitions

[0050] “Fusion polypeptide” or “fusion protein” refers to a proteinhaving at least two heterologous Mycobacterium sp. polypeptidescovalently linked, either directly or via an amino acid linker. Thepolypeptides forming the fusion protein are typically linked C-terminusto N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Thepolypeptides of the fusion protein can be in any order. This term alsorefers to conservatively modified variants, polymorphic variants,alleles, mutants, subsequences, interspecies homologs, and immunogenicfragments of the antigens that make up the fusion protein. Mycobacteriumtuberculosis antigens are described in Cole et al., Nature 393:537(1998), which discloses the entire Mycobacterium tuberculosis genome.The complete sequence of Mycobacterium tuberculosis can also be found athttp://www.sanger.ac.uk and at http://www.pasteur.fr/mycdb/ (MycDB).Antigens from other Mycobacterium species that correspond to M.tuberculosis antigens can be identified, e.g., using sequence comparisonalgorithms, as described herein, or other methods known to those ofskill in the art, e.g., hybridization assays and antibody bindingassays. Fusion proteins of the invention can also comprise additionalcopies of a component antigen or immunogenic fragment thereof.

[0051] A polynucleotide sequence comprising a fusion protein of theinvention hybridizes under stringent conditions to at least twonucleotide sequences, each encoding an antigen polypeptide selected fromthe group consisting of MTB39 or an immunogenic fragment thereof,mutated MTB32A or an immunogenic fragment thereof, and 85B or animmunogenic fragment thereof. The polynucleotide sequences encoding theindividual antigens of the fusion polypeptide therefore includeconservatively modified variants, polymorphic variants, alleles,mutants, subsequences, immunogenic fragments, and interspecies homologsof MTB39, MTB32A, and 85B. The polynucleotide sequence encoding theindividual polypeptides of the fusion protein can be in any order.

[0052] In some embodiments, the individual polypeptides of the fusionprotein are in order (N- to C-terminus) from large to small. Largeantigens are approximately 30 to 150 kD in size, medium antigens areapproximately 10 to 30 kD in size, and small antigens are approximatelyless than 10 kD in size. The sequence encoding the individualpolypeptide may be as small as, e.g., an immunogenic fragment such as anindividual CTL epitope encoding about 8 to 9 amino acids, or, e.g., anHTL or B cell epitope. The fragment may also include multiple epitopes.The immunogenic fragment may also represent a larger part of the antigensequence, e.g., about 50% or more of MTB39, 85B, and MTB32A, e.g., theN- and C-terminal portions of MTB32A.

[0053] A fusion polypeptide of the invention specifically binds toantibodies raised against at least two antigen polypeptides, whereineach antigen polypeptide is selected from the group consisting of MTB39or an immunogenic portion or fragment thereof, mutated MTB32A or animmunogenic portion thereof, and 85B or an immunogenic portion therof.The antibodies can be polyclonal or monoclonal. Optionally, the fusionpolypeptide specifically binds to antibodies raised against the fusionjunction of the antigens, which antibodies do not bind to the antigensindividually, i.e., when they are not part of a fusion protein. Thefusion polypeptides optionally comprise additional polypeptides, e.g.,three, four, five, six, or more polypeptides, up to about 25polypeptides, optionally heterologous polypeptides or repeatedhomologous polypeptides, fused to the at least two heterologousantigens. The additional polypeptides of the fusion protein areoptionally derived from Mycobacterium as well as other sources, such asother bacterial, viral, or invertebrate, vertebrate, or mammaliansources. The individual polypeptides of the fusion protein can be in anyorder. As described herein, the fusion protein can also be linked toother molecules, including additional polypeptides. The compositions ofthe invention can also comprise additional polypeptides that areunlinked to the fusion proteins of the invention. These additionalpolypeptides may be heterologous or homologous polypeptides.

[0054] The term “fused” refers to the covalent linkage between twopolypeptides in a fusion protein. The polypeptides are typically joinedvia a peptide bond, either directly to each other or via an amino acidlinker. Optionally, the peptides can be joined via non-peptide covalentlinkages known to those of skill in the art.

[0055] “FL” refers to full-length, i.e., a polypeptide that is the samelength as the wild-type polypeptide. In some embodiment, the FL versionis the mature version, that is, the secreted, full length form lackingthe signal sequence.

[0056] The term “immunogenic fragment thereof” refers to a polypeptidecomprising an epitope that is recognized by cytotoxic T lymphocytes,helper T lymphocytes or B cells.

[0057] An amount of a composition, nucleic acid, or fusion protein thatelicits an immune response is an “immunogenically” or “immunologically”“effective amount” of the composition, nucleic acid or polypeptide.

[0058] MTB32AMutSA is a mutated version of wild-type MTB32A (Ra35FL orRa35 mature). The sequence of wild-type RA35 is disclosed as SEQ IDNO:17 (cDNA) and SEQ ID NO:79 (protein) in the U.S. patent applicationSer. Nos. 08/523,436, 08/523,435, 08/658,800, 08/659,683, 08/818,112,09/056,556, and 08/818,111 and in the WO97/09428 and WO97/09429applications, see also Skeiky et al, Infection and Immunity 67:3998-4007(1999). The term mutated MTB32, mutated MTB32A, MTB32AMutSA orMTB32MutSA includes MTB32A amino acid sequences in which any one of thethree amino acids at the active site triad (His, Asp, Ser, amino acidpositions 182-184 of the wild type molecule), e.g., the serine residueat amino acid position 183 in wild-type MTB32A, has been changed toanother amino acid (e.g., to alanine, Ra35FLMutSA, see, e.g., thesequence comparison of wild type and mutated MTB32 in FIG. 5).

[0059] MTB39 (TbH9), the sequence of which is disclosed as SEQ ID NO:106(cDNA full length) and SEQ ID NO:107 (protein full length) in the U.S.patent application Ser. Nos. 08/658,800, 08/659,683, 08/818,112, and08/818,111 and in the WO97/09428 and WO97/09429 applications. Thesequence is also disclosed as SEQ ID NO:33 (DNA) and SEQ ID NO:91 (aminoacid) in U.S. patent application Ser. No. 09/056,559.

[0060] MTB72F (Ra12-TbH9-Ra35), the sequence of which is disclosed asSEQ ID NO:1 (DNA) and SEQ ID NO:2 (protein) in the U.S. patentapplication Ser. Nos. 09/223,040, 09/223,040, and in the PCT/US99/07717application.

[0061] 85 complex antigen, e.g., 85b antigen from M. bovis, the sequenceof which is disclosed in Content et al., Infect. & Immunol. 59:3205-3212(1991).

[0062] The term “Mycobacterium species of the tuberculosis complex”includes those species traditionally considered as causing the diseasetuberculosis, as well as Mycobacterium environmental and opportunisticspecies that cause tuberculosis and lung disease in immune compromisedpatients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis,or M. africanum, BCG, M. avium, M. intracellulare, M. celatum, M.genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, Mfortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles ofInternal Medicine, volume 1, pp. 1004-1014 and 1019-1023 (14^(th) ed.,Fauci et al., eds., 1998).

[0063] An adjuvant refers to the components in a vaccine or therapeuticcomposition that increase the specific immune response to the antigen(see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)).Adjuvants induce immune responses of the Th1-type and Th-2 typeresponse. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend tofavor the induction of cell-mediated immune response to an administeredantigen, while Th-2 type cytokines (e.g., IL-4, IL-5, IL-6, IL-10 andTNF-β) tend to favor the induction of humoral immune responses.

[0064] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0065] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0066] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0067] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0068] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0069] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0070] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0071] The following eight groups each contain amino acids that areconservative substitutions for one another:

[0072] 1) Alanine (A), Glycine (G);

[0073] 2) Aspartic acid (D), Glutamic acid (E);

[0074] 3) Asparagine (N), Glutamine (Q);

[0075] 4) Arginine (R), Lysine (K);

[0076] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0077] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0078] 7) Serine (S), Threonine (T); and

[0079] 8) Cysteine (C), Methionine (M)

[0080] (see, e.g., Creighton, Proteins (1984)).

[0081] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0082] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0083] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0084] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0085] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0086] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0087] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

[0088] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy(1985)). Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptidesof this invention. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0089] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to fusion proteins can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with fusion protein and not with individualcomponents of the fusion proteins. This selection may be achieved bysubtracting out antibodies that cross-react with the individualantigens. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

[0090] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes an individual antigen or a portionthereof) or may comprise a variant of such a sequence. Polynucleotidevariants may contain one or more substitutions, additions, deletionsand/or insertions such that the biological activity of the encodedfusion polypeptide is not diminished, relative to a fusion polypeptidecomprising native antigens. Variants preferably exhibit at least about70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native polypeptide or a portion thereof.

[0091] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length.

[0092] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0093] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 25 to 500, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wis. GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds.

[0094] 1995 supplement)).

[0095] One example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0096] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0097] Polynucleotide Compositions

[0098] As used herein, the terms “DNA segment” and “polynucleotide”refer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

[0099] As will be understood by those skilled in the art, the DNAsegments of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

[0100] The terms “isolated,” “purified,” or “biologically pure”therefore refer to material that is substantially or essentially freefrom components that normally accompany it as found in its native state.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude other isolated proteins, genes, or coding regions lateradded to the composition by the hand of man. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. An isolated nucleic acid isseparated from other open reading frames that flank the gene and encodeproteins other than the gene.

[0101] As will be recognized by the skilled artisan, polynucleotides maybe single-stranded (coding or antisense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

[0102] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes a Mycobacterium antigen or a portionthereof) or may comprise a variant, or a biological or antigenicfunctional equivalent of such a sequence. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions, as further described below, preferably such that theimmunogenicity of the encoded polypeptide is not diminished, relative toa native tumor protein. The effect on the immunogenicity of the encodedpolypeptide may generally be assessed as described herein. The term“variants” also encompasses homologous genes of xenogenic origin.

[0103] In additional embodiments, the present invention providesisolated polynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

[0104] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrative DNAsegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

[0105] Moreover, it will be appreciated by those of ordinary skill inthe art that, as a result of the degeneracy of the genetic code, thereare many nucleotide sequences that encode a MTB32Mut-39F or MTB 102Fpolypeptide as described herein. Some of these polynucleotides bearminimal homology to the nucleotide sequence of any native gene.Nonetheless, polynucleotides that vary due to differences in codon usageare specifically contemplated by the present invention, for examplepolynucleotides that are optimized for human and/or primate codonselection. Further, alleles of the genes comprising the polynucleotidesequences provided herein are within the scope of the present invention.Alleles are endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

[0106] Polynucleotide Identification and Characterization

[0107] Polynucleotides encoding MTB32Mut-39F or MTB102F may beidentified, prepared and/or manipulated using any of a variety of wellestablished techniques. For example, a polynucleotide encodingMTB32Mut-39F or MTB102F may be identified, as described in more detailbelow, by screening a microarray of cDNAs for tumor-associatedexpression (i.e., expression that is at least two fold greater in atumor than in normal tissue, as determined using a representative assayprovided herein). Such screens may be performed, for example, using aSynteni microarray (Palo Alto, Calif.) according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Natl.Acad. Sci. USA 93:10614-10619 (1996) and Heller et al., Proc. Natl.Acad. Sci. USA 94:2150-2155 (1997)). Alternatively, polynucleotides maybe amplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as M. tuberculosis cells. Such polynucleotidesmay be amplified via polymerase chain reaction (PCR). For this approach,sequence-specific primers may be designed based on the sequencesprovided herein, and may be purchased or synthesized.

[0108] An amplified portion of a polynucleotide of the present inventionmay be used to isolate a full length gene from a suitable library (e.g.,a M. tuberculosis cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

[0109] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual (1989)). Hybridizingcolonies or plaques are selected and expanded, and the DNA is isolatedfor further analysis. cDNA clones may be analyzed to determine theamount of additional sequence by, for example, PCR using a primer fromthe partial sequence and a primer from the vector. Restriction maps andpartial sequences may be generated to identify one or more overlappingclones. The complete sequence may then be determined using standardtechniques, which may involve generating a series of deletion clones.The resulting overlapping sequences can then assembled into a singlecontiguous sequence. A full length cDNA molecule can be generated byligating suitable fragments, using well known techniques.

[0110] Alternatively, there are numerous amplification techniques forobtaining a full length coding sequence from a partial cDNA sequence.Within such techniques, amplification is generally performed via PCR.Any of a variety of commercially available kits may be used to performthe amplification step. Primers may be designed using, for example,software well known in the art. Primers are preferably 22-30 nucleotidesin length, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

[0111] One such amplification technique is inverse PCR (see Triglia etal., Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60 (1991)). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

[0112] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

[0113] Polynucleotide Expression in Host Cells

[0114] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode MTB32Mut-39F or MTB102F, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a MTB32Mut-39F or MTB 102Fpolypeptide in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences that encode substantially the sameor a functionally equivalent amino acid sequence may be produced andthese sequences may be used to clone and express a given polypeptide.

[0115] As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

[0116] Moreover, the polynucleotide sequences of the present inventioncan be engineered using methods generally known in the art in order toalter polypeptide encoding sequences for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the gene product. For example, DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

[0117] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

[0118] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980),Horn et al., Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of a polypeptide, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge et al., Science 269:202-204 (1995)) andautomated synthesis may be achieved, for example, using the ABI 431 APeptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0119] A newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,Proteins, Structures and Molecular Principles (1983)) or othercomparable techniques available in the art. The composition of thesynthetic peptides may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure). Additionally, the amino acidsequence of a polypeptide, or any part thereof, may be altered duringdirect synthesis and/or combined using chemical methods with sequencesfrom other proteins, or any part thereof, to produce a variantpolypeptide.

[0120] In order to express a desired polypeptide, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods which are well known to thoseskilled in the art may be used to construct expression vectorscontaining sequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described in Sambrooket al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel etal., Current Protocols in Molecular Biology (1989).

[0121] A variety of expression vector/host systems may be utilized tocontain and express polynucleotide sequences. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0122] The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

[0123] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509(1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also beused to express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0124] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al., Methods Enzymol. 153:516-544 (1987).

[0125] In cases where plant expression vectors are used, the expressionof sequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw HillYearbook of Science and Technology pp. 191-196 (1992)).

[0126] An insect system may also be used to express a polypeptide ofinterest. For example, in one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The sequences encoding the polypeptide may be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe polypeptide-encoding sequence will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which the polypeptide ofinterest may be expressed (Engelhard et al., Proc. Natl. Acad. Sci.U.S.A. 91:3224-3227 (1994)).

[0127] In mammalian host cells, a number of viral-based expressionsystems are generally available. For example, in cases where anadenovirus is used as an expression vector, sequences encoding apolypeptide of interest may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan &Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0128] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding a polypeptide of interest.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

[0129] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0130] For long-term, high-yield production of recombinant proteins,stable expression is generally preferred. For example, cell lines whichstably express a polynucleotide of interest may be transformed usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

[0131] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-32(1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell22:817-23 (1990)) genes which can be employed in tk.sup.- or aprt.sup.-cells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate (Wigler et al., Proc. Natl.Acad. Sci. US.A. 77:3567-70 (1980)); npt, which confers resistance tothe aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol.Biol. 150:1-14 (1981)); and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51(1988)). Recently, the use of visible markers has gained popularity withsuch markers as anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0132] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0133] Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

[0134] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide may bepreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton et al., Serological Methods, a Laboratory Manual(1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0135] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0136] Host cells transformed with a polynucleotide sequence of interestmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides ofthe invention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porathet al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll et al., DNA Cell Biol. 12:441-453 (1993)).

[0137] In addition to recombinant production methods, MTB32Mut-39F orMTB102F polypeptides of the invention, and fragments thereof, may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Alternatively, various fragments maybe chemically synthesized separately and combined using chemical methodsto produce the full length molecule.

[0138] In Vivo Polynucleotide Delivery Techniques

[0139] In additional embodiments, genetic constructs comprising one ormore of the polynucleotides encoding mutated MTB32Mut-39F or animmunogenic fragment thereof; or MTB102F or an immunogenic fragmentthereof are introduced into cells in vivo. This may be achieved usingany of a variety or well known approaches, several of which are outlinedbelow for the purpose of illustration.

[0140] 1. Adenovirus

[0141] One of the preferred methods for in vivo delivery of one or morenucleic acid sequences involves the use of an adenovirus expressionvector. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to express a polynucleotide that hasbeen cloned therein in a sense or antisense orientation. Of course, inthe context of an antisense construct, expression does not require thatthe gene product be synthesized.

[0142] The expression vector comprises a genetically engineered form ofan adenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus &Horwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

[0143] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0144] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0145] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones & Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the E1, the D3 or bothregions (Graham & Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

[0146] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

[0147] Recently, Racher et al. (1995) disclosed improved methods forculturing 293 cells and propagating adenovirus. In one format, naturalcell aggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

[0148] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

[0149] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

[0150] Adenovirus is easy to grow and manipulate and exhibits broad hostrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

[0151] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Recently, animalstudies suggested that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet & Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz & Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

[0152] 2. Retroviruses

[0153] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0154] In order to construct a retroviral vector, a nucleic acidencoding one or more oligonucleotide or polynucleotide sequences ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and packaging components is constructed (Mannet al., 1983). When a recombinant plasmid containing a cDNA, togetherwith the retroviral LTR and packaging sequences is introduced into thiscell line (by calcium phosphate precipitation for example), thepackaging sequence allows the RNA transcript of the recombinant plasmidto be packaged into viral particles, which are then secreted into theculture media (Nicolas & Rubenstein, 1988; Temin, 1986; Mann et al.,1983). The media containing the recombinant retroviruses is thencollected, optionally concentrated, and used for gene transfer.Retroviral vectors are able to infect a broad variety of cell types.However, integration and stable expression require the division of hostcells (Paskind et al., 1975).

[0155] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of lactoseresidues to the viral envelope. This modification could permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

[0156] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

[0157] 3. Adeno-Associated Viruses

[0158] AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin,1988).

[0159] The AAV DNA is approximately 4.7 kilobases long. It contains twoopen reading frames and is flanked by two ITRs. There are two majorgenes in the AAV genome: rep and cap. The rep gene codes for proteinsresponsible for viral replications, whereas cap codes for capsid proteinVP1-3. Each ITR forms a T-shaped hairpin structure. These terminalrepeats are the only essential cis components of the AAV for chromosomalintegration. Therefore, the AAV can be used as a vector with all viralcoding sequences removed and replaced by the cassette of genes fordelivery. Three viral promoters have been identified and named p5, p19,and p40, according to their map position. Transcription from p5 and p19results in production of rep proteins, and transcription from p40produces the capsid proteins (Hermonat & Muzyczka, 1984).

[0160] There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

[0161] AAV is also a good choice of delivery vehicles due to its safety.There is a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

[0162] 4. Other Viral Vectors as Expression Constructs

[0163] Other viral vectors may be employed as expression constructs inthe present invention for the delivery of oligonucleotide orpolynucleotide sequences to a host cell. Vectors derived from virusessuch as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988),lentiviruses, polio viruses and herpes viruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al.,1990).

[0164] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

[0165] 5. Non-Viral Vectors

[0166] In order to effect expression of the oligonucleotide orpolynucleotide sequences of the present invention, the expressionconstruct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. As described above, one preferred mechanism for deliveryis via viral infection where the expression construct is encapsulated inan infectious viral particle.

[0167] Once the expression construct has been delivered into the cellthe nucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

[0168] In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty & Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

[0169] Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

[0170] Selected organs including the liver, skin, and muscle tissue ofrats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin etal., 1991). This may require surgical exposure of the tissue or cells,to eliminate any intervening tissue between the gun and the targetorgan, i.e., ex vivo treatment. Again, DNA encoding a particular genemay be delivered via this method and still be incorporated by thepresent invention.

[0171] Polypeptide Compositions

[0172] The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

[0173] Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of aMycobacterium sp. protein is a portion that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the fill length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow & Lane, Antibodies: A Laboratory Manual(1988). For example, a polypeptide may be immobilized on a solid supportand contacted with patient sera to allow binding of antibodies withinthe sera to the immobilized polypeptide. Unbound sera may then beremoved and bound antibodies detected using, for example, ¹²⁵I-labeledProtein A.

[0174] Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

[0175] Polypeptides of the invention, immunogenic fragments thereof, andother variants having less than about 100 amino acids, and generallyless than about 50 amino acids, may also be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. For example, such polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.85:2149-2146 (1963). Equipment for automated synthesis of polypeptidesis commercially available from suppliers such as Perkin Elmer/AppliedBioSystems Division (Foster City, Calif.), and may be operated accordingto the manufacturer's instructions.

[0176] Within certain specific embodiments, a polypeptide may be afusion protein that comprises multiple polypeptides as described herein,or that comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

[0177] Fusion proteins may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusion proteinis expressed as a recombinant protein, allowing the production ofincreased levels, relative to a non-fused protein, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion protein that retains the biological activity ofboth component polypeptides.

[0178] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

[0179] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

[0180] Fusion proteins are also provided. Such proteins comprise apolypeptide as described herein together with an unrelated immunogenicprotein. Preferably the immunogenic protein is capable of eliciting arecall response. Examples of such proteins include tetanus, tuberculosisand hepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med.336:86-91 (1997)).

[0181] Within preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

[0182] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terminalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292 (1986)). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli CLYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0183] In general, polypeptides (including fusion proteins) andpolynucleotides as described herein are isolated. An “isolated”polypeptide or polynucleotide is one that is removed from its originalenvironment. For example, a naturally-occurring protein is isolated ifit is separated from some or all of the coexisting materials in thenatural system. Preferably, such polypeptides are at least about 90%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure. A polynucleotide is considered to be isolated if,for example, it is cloned into a vector that is not a part of thenatural environment.

[0184] T Cells

[0185] Immunotherapeutic compositions may also, or alternatively,comprise T cells specific for MTB32Mut-39F or an immunogenic fragmentthereof and MTB102F or an immunogenic fragment thereof. Such cells maygenerally be prepared in vitro or ex vivo, using standard procedures.For example, T cells may be isolated from bone marrow, peripheral blood,or a fraction of bone marrow or peripheral blood of a patient, using acommercially available cell separation system, such as the Isolex™System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; seealso U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO91/16116 and WO 92/07243). Alternatively, T cells may be derived fromrelated or unrelated humans, non-human mammals, cell lines or cultures.

[0186] T cells may be stimulated with MTB32Mut-39F or MTB102F, apolynucleotide encoding MTB32Mut-39F or MTB102F, and/or an antigenpresenting cell (APC) that presents an antigenic portion of MTB32Mut-39For MTB102F. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor MTB32Mut-39F or MTB102F. Preferably, the MTB32Mut-39F or MTB102F orpolynucleotide encoding MTB32Mut-39F or MTB102F is present within adelivery vehicle, such as a microsphere, to facilitate the generation ofMTB32Mut-39F or MTB102F specific T cells.

[0187] T cells are considered to be specific for MTB32Mut-39F or MTB102Fif the T cells specifically proliferate, secrete cytokines or killtarget cells coated with MTB32Mut-39F or an immunogenic fragmentthereof; or MTB102F or an immunogenic fragment thereof; or expressing agene encoding MTB32Mut-39F or MTB102F. T cell specificity may beevaluated using any of a variety of standard techniques. For example,within a chromium release assay or proliferation assay, a stimulationindex of more than two fold increase in lysis and/or proliferation,compared to negative controls, indicates T cell specificity. Such assaysmay be performed, for example, as described in Chen et al., Cancer Res.54:1065-1070 (1994)). Alternatively, detection of the proliferation of Tcells may be accomplished by a variety of known techniques. For example,T cell proliferation can be detected by measuring an increased rate ofDNA synthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Contact with a polypeptide of the invention (100ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days shouldresult in at least a two fold increase in proliferation of the T cells.Contact as described above for 2-3 hours should result in activation ofthe T cells, as measured using standard cytokine assays in which a twofold increase in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a MTB32Mut-39F or MTB102F, polynucleotide encodingMTB32Mut-39F or MTB102F or MTB32Mut-39F or MTB 102F presenting APC maybe CD4⁺ and/or CD8⁺. MTB32Mut-39F or MTB102F specific T cells may beexpanded using standard techniques. Within preferred embodiments, the Tcells are derived from a patient, a related donor or an unrelated donor,and are administered to the patient following stimulation and expansion.

[0188] For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferatein response to MTB32Mut-39F or MTB102F, a polynucleotide encodingMTB32Mut-39F or MTB102F or APC presenting antigenic peptides fromMTB32Mut-39F or MTB 102F can be expanded in number either in vitro or invivo. Proliferation of such T cells in vitro may be accomplished in avariety of ways. For example, the T cells can be re-exposed toMTB32Mut-39F or MTB102F, or a short peptide corresponding to animmunogenic portion of such MTB32Mut-39F or MTB102F, with or without theaddition of T cell growth factors, such as interleukin-2, and/orstimulator cells that synthesize the polypeptide. Alternatively, one ormore T cells that proliferate in the presence of MTB32Mut-39F or MTB102Fcan be expanded in number by cloning. Methods for cloning cells are wellknown in the art, and include limiting dilution.

[0189] Pharmaceutical Compositions

[0190] In additional embodiments, the present invention concernsformulation of one or more of the polynucleotide, polypeptide, T-celland/or antibody compositions disclosed herein inpharmaceutically-acceptable or physiologically-acceptable solutions foradministration to a cell or an animal, either alone, or in combinationwith one or more other modalities of therapy. Such compositions are alsouseful for diagnostic uses.

[0191] It will also be understood that, if desired, the nucleic acidsegment, RNA, DNA compositions that express a MTB32Mut-39F or MTB102F asdisclosed herein may be administered in combination with other agents aswell, such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

[0192] Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

[0193] 1. Oral Delivery

[0194] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0195] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitzet al., 1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

[0196] Typically, these formulations may contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0197] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. For example, a mouthwash maybe prepared incorporating the active ingredient in the required amountin an appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

[0198] 2. Injectable Delivery

[0199] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0200] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0201] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion (see, e.g.,Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, and the general safety and puritystandards as required by FDA Office of Biologics standards.

[0202] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0203] The compositions disclosed herein may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

[0204] As used herein, “carrier” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0205] The phrase “pharmaceutically-acceptable” refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to a human. The preparation of anaqueous composition that contains a protein as an active ingredient iswell understood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

[0206] 3. Nasal Delivery

[0207] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212 (each specifically incorporated herein by reference in itsentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

[0208] 4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

[0209] In certain embodiments, the inventors contemplate the use ofliposomes, nanocapsules, microparticles, microspheres, lipid particles,vesicles, and the like, for the introduction of the compositions of thepresent invention into suitable host cells. In particular, thecompositions of the present invention may be formulated for deliveryeither encapsulated in a lipid particle, a liposome, a vesicle, ananosphere, or a nanoparticle or the like.

[0210] Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

[0211] Liposomes have been used successfully with a number of cell typesthat are normally resistant to transfection by other proceduresincluding T cell suspensions, primary hepatocyte cultures and PC 12cells (Renneisen et al., 1990; Muller et al., 1990). In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs (Heath & Martin, 1986; Heath et al., 1986;Balazsovits et al., 1989; Fresta & Puglisi, 1996), radiotherapeuticagents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumiet al., 1990b), viruses (Faller & Baltimore, 1984), transcriptionfactors and allosteric effectors (Nicolau & Gersonde, 1979) into avariety of cultured cell lines and animals. In addition, severalsuccessful clinical trails examining the effectiveness ofliposome-mediated drug delivery have been completed (Lopez-Berestein etal., 1985a; 1985b; Coune, 1988; Sculier et al., 1988). Furthermore,several studies suggest that the use of liposomes is not associated withautoimmune responses, toxicity or gonadal localization after systemicdelivery (Mori & Fukatsu, 1992).

[0212] Liposomes are formed from phospholipids that are dispersed in anaqueous medium and spontaneously form multilamellar concentric bilayervesicles (also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

[0213] Liposomes bear resemblance to cellular membranes and arecontemplated for use in connection with the present invention ascarriers for the peptide compositions. They are widely suitable as bothwater- and lipid-soluble substances can be entrapped, i.e. in theaqueous spaces and within the bilayer itself, respectively. It ispossible that the drug-bearing liposomes may even be employed forsite-specific delivery of active agents by selectively modifying theliposomal formulation.

[0214] In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

[0215] In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

[0216] The ability to trap solutes varies between different types ofliposomes. For example, MLVs are moderately efficient at trappingsolutes, but SUVs are extremely inefficient. SUVs offer the advantage ofhomogeneity and reproducibility in size distribution, however, and acompromise between size and trapping efficiency is offered by largeunilamellar vesicles (LUVs). These are prepared by ether evaporation andare three to four times more efficient at solute entrapment than MLVs.

[0217] In addition to liposome characteristics, an important determinantin entrapping compounds is the physicochemical properties of thecompound itself. Polar compounds are trapped in the aqueous spaces andnonpolar compounds bind to the lipid bilayer of the vesicle. Polarcompounds are released through permeation or when the bilayer is broken,but nonpolar compounds remain affiliated with the bilayer unless it isdisrupted by temperature or exposure to lipoproteins. Both types showmaximum efflux rates at the phase transition temperature.

[0218] Liposomes interact with cells via four different mechanisms:endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

[0219] The fate and disposition of intravenously injected liposomesdepend on their physical properties, such as size, fluidity, and surfacecharge. They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

[0220] Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

[0221] Alternatively, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (Henry-Michelland et al., 1987;Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention. Such particles may be are easily made, asdescribed (Couvreur et al, 1980; 1988; zur Muhlen et al., 1998; Zambauxet al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684,specifically incorporated herein by reference in its entirety).

[0222] Vaccines

[0223] In certain preferred embodiments of the present invention,vaccines are provided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, Powell & Newman,eds., Vaccine Design (the subunit and adjuvant approach) (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens may be present, either incorporated into a fusionpolypeptide or as a separate compound, within the composition orvaccine.

[0224] Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. As noted above, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198 (1998), and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface or secretessuch an epitope. In a preferred embodiment, the DNA may be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321 (1989); Flexner et al., Ann. N.Y. Acad. Sci.569:86-103 (1989); Flexner et al., Vaccine 8:17-21 (1990); U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627 (1988); Rosenfeld et al., Science 252:431-434(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502 (1993);Guzman et al., Circulation 88:2838-2848 (1993); and Guzman et al., Cir.Res. 73:1202-1207 (1993). Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749 (1993) and reviewed by Cohen, Science259:1691-1692 (1993). The uptake of naked DNA may be increased bycoating the DNA onto biodegradable beads, which are efficientlytransported into the cells. It will be apparent that a vaccine maycomprise both a polynucleotide and a polypeptide component. Suchvaccines may provide for an enhanced immune response.

[0225] It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

[0226] While any suitable carrier known to those of ordinary skill inthe art may be employed in the vaccine compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

[0227] Such compositions may also comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, bacteriostats, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminumhydroxide), solutes that render the formulation isotonic, hypotonic orweakly hypertonic with the blood of a recipient, suspending agents,thickening agents and/or preservatives. Alternatively, compositions ofthe present invention may be formulated as a lyophilizate. Compounds mayalso be encapsulated within liposomes using well known technology.

[0228] Any of a variety of immunostimulants may be employed in thevaccines of this invention. For example, an adjuvant may be included.Most adjuvants contain a substance designed to protect the antigen fromrapid catabolism, such as aluminum hydroxide or mineral oil, and astimulator of immune responses, such as lipid A, Bortadella pertussis orMycobacterium species or Mycobacterium derived proteins. For example,delipidated, deglycolipidated M. vaccae (“pVac”) can be used. In anotherembodiment, BCG is used as an adjuvant. In addition, the vaccine can beadministered to a subject previously exposed to BCG. Suitable adjuvantsare commercially available as, for example, Freund's Incomplete Adjuvantand Complete Adjuvant (Difco Laboratories, Detroit, Mich.); MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.); CWS, MPL, CpG, TDM,Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0229] Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann & Coffman, Ann. Rev.Immunol. 7:145-173 (1989).

[0230] Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352 (1996). Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

[0231] Alternatively the saponin formulations may be combined withvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

[0232] In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion (optionally withsqualene) is described in WO 95/17210. CpG is optionally a component ofthese adjuvant systems. See also EP 735898 B1.

[0233] Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion (optionallyusing squalene) and tocopherol.

[0234] Other preferred adjuvants include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton,Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such asthose described in pending U.S. patent application Ser. Nos. 08/853,826and 09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

[0235] Other preferred adjuvants include adjuvant molecules of thegeneral formula (I): HO(CH₂CH₂O)_(n)-A-R,

[0236] wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orphenyl C₁₋₅₀ alkyl.

[0237] One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

[0238] The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

[0239] Any vaccine provided herein may be prepared using well knownmethods that result in a combination of antigen, immune responseenhancer and a suitable carrier or excipient. The compositions describedherein may be administered as part of a sustained release formulation(i.e., a formulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438 (1996)) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.Vaccines can be administered in a prime and boost combination, e.g.,priming with a nucleic acid encoding a fusion protein of the inventionand then later boosting with a dose of the fusion protein, oralternatively priming with a fusion protein of the invention and thenlater boosting with a dose of the nucleic acid encoding the fusionprotein.

[0240] Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of active component release. Such carriersinclude microparticles of poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose, dextran and the like. Other delayed-releasecarriers include supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

[0241] Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of aMTB32Mut-39F or MTB102F-specific immune response that targets tumorcells. Delivery vehicles include antigen presenting cells (APCs), suchas dendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

[0242] Certain preferred embodiments of the present invention usedendritic cells or progenitors thereof as antigen-presenting cells.Dendritic cells are highly potent APCs (Banchereau & Steinman, Nature392:245-251 (1998)) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman & Levy, Ann. Rev. Med. 50:507-529(1999)). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600 (1998)).

[0243] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, tumor-infiltrating cells, peritumoraltissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cordblood or any other suitable tissue or fluid. For example, dendriticcells may be differentiated ex vivo by adding a combination of cytokinessuch as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytesharvested from peripheral blood. Alternatively, CD34 positive cellsharvested from peripheral blood, umbilical cord blood or bone marrow maybe differentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

[0244] Dendritic cells are conveniently categorized as “immature” and“mature” cells, which allows a simple way to discriminate between twowell characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

[0245] APCs may generally be transfected with a polynucleotide encodingMTB32Mut-39F or MTB102F (or portion or other variant thereof) such thatthe MTB32Mut-39F or an immunogenic fragment thereof; or MTB102F or animmunogenic portion thereof, is expressed on the cell surface. Suchtransfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in WO 97/24447, or the gene gun approach described byMahvi et al., Immunology and Cell Biology 75:456-460 (1997). Antigenloading of dendritic cells may be achieved by incubating dendritic cellsor progenitor cells with MTB32Mut-39F or MTB102F, DNA (naked or within aplasmid vector) or RNA; or with antigen-expressing recombinant bacteriumor viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, MTB32Mut-39F or MTB102F may be covalently conjugatedto an immunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofMTB32Mut-39F or MTB102F.

[0246] Vaccines and pharmaceutical compositions may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are preferably hermetically sealed to preserve sterilityof the formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

[0247] Diagnostic Kits

[0248] The present invention further provides kits for use within any ofthe above diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

[0249] Alternatively, a kit may be designed to detect the level of mRNAencoding a protein in a biological sample. Such kits generally compriseat least one oligonucleotide probe or primer, as described above, thathybridizes to a polynucleotide encoding a protein. Such anoligonucleotide may be used, for example, within a PCR or hybridizationassay. Additional components that may be present within such kitsinclude a second oligonucleotide and/or a diagnostic reagent orcontainer to facilitate the detection of a polynucleotide encoding aprotein of the invention.

[0250] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0251] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

[0252] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

Example 1 Construction of Vector for MTB32MutSA-MTB39F and MTB102FFusion Polypeptides

[0253] Expression of the full-length sequences of the mature or fulllength, secreted form of Ra35 (MTB32A) in E. coli has been difficult.The expressed product was only visible after immunoblotting with apolyclonal rabbit anti-Ra35 antibody indicative of low levels of proteinexpression. Even then, multiple specific species (bands) were detectedindicative of auto-catalytic breakdown (degradation) of the recombinantantigen. This result was presumed to be due to the expression of Ra35FLin E. coli as a biologically active form.

[0254] It has been previously shown that it was possible to expressRa35FL as two overlapping halves comprising the N-terminal (Ra35N-term,called Ra35) and C-term halves (Ra35C-term called Ra12). To enhance andstabilize the expression of the whole Ra35 molecule, a single pointmutation was introduced at one of the residues within the active-sitetriad (T to G, resulting in substitution of Ser to Ala). Thismutagenized form of MTB32A can now be easily expressed at high levels ina stable form.

[0255] To increase fusion protein expression level, techniques wellknown in the art were used to generate a fusion protein, mutatedMTB32-39F, including both MTB32MutSA and MTB39. MTB32-39F is 723 aminoacid polypeptide which includes amino acids 1-330 from Ra35FLMutSA andamino acids 1-391 of MTB39. MTB32MutSA-39F is stable, is expressed athigh levels, and is highly immunogenic in animal studies.

Example 2 MTB102F

[0256] MTB102F was created by adding a third antigen, MTB 85B, to the Cterminus of MTB32MutSA-MTB39F. MTB85B is a 287 amino acid sequencederived from Mycobacterium bovis (see, e.g., Content et al., Infect. &Immunol. 59:3205-3212 (1991)).

What is claimed is:
 1. An isolated nucleic acid encoding a fusionpolypeptide comprising a MTB32A antigen and a MTB39 antigen from aMycobacterium species of the tuberculosis complex, wherein said nucleicacid hybridizes under highly stringent conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:1 or a complement thereof,and wherein the MTB32A antigen has a mutation at amino acid position 183as compared to wild type MTB32A.
 2. The nucleic acid of claim 1, whereinthe mutation is a serine to alanine mutation. 3 The nucleic acid ofclaim 1, comprising a nucleotide sequence SEQ ID NO:1.
 4. The nucleicacid of claim 1, encoding an amino acid sequence of SEQ ID NO:2.
 5. Thenucleic acid of claim 1, comprising a nucleotide sequence SEQ ID NO:3.6. The nucleic acid of claim 1, encoding an amino acid sequence of SEQID NO:4.
 7. The nucleic acid of claim 1, further encoding an MTB85Bantigen from a Mycobacterium species of the tuberculosis complex or animmunogenic fragment thereof.
 8. The nucleic acid of claim 7, whereinthe MTB85B antigen is from M bovis and the MTB32A antigen and the MTB39antigen are from M. tuberculosis.
 9. The nucleic acid of claim 1,wherein the Mycobacterium is Mycobacterium tuberculosis.
 10. Anexpression vector comprising the nucleic acid of claim
 1. 11. A hostcell comprising the expression vector of claim
 10. 12. The host cell ofclaim 11, wherein the host cell is selected from the group consisting ofE. coli, yeast, and mammalian cells.
 13. A composition comprising anucleic acid according to claim 1 and a physiologically acceptablecarrier.
 14. The composition of claim 13, wherein the fusion polypeptideencoded by the nucleic acid further comprises an NS1 antigen or animmunogenic fragment therof.
 15. The composition of claim 13, whereinthe Mycobacterium species is Mycobacterium tuberculosis.
 16. An isolatedfusion protein encoded by the nucleic acid of claim
 1. 17. A compositioncomprising a fusion protein of claim 16 and a physiologically acceptablecarrier.
 18. A composition of claim 17, comprising a non-specific immuneresponse enhancer.
 19. The composition of claim 18, wherein thenonspecific immune response enhancer is an adjuvant.
 20. The compositionof claim 19, wherein the adjuvant comprises QS21 and MPL.
 21. Thecomposition of claim 19, wherein the adjuvant comprises QS21, MPL, andCpG.
 22. The composition of claim 19, wherein the adjuvant is selectedfrom the group consisting of ENHANZYN, MPL, 3D-MPL, IFA, QS21, CWS, TDM,AGP, CpG, Leif, saponin, and saponin mimetics.
 23. The composition ofclaim 17, further comprising BCG or pVac.
 24. The composition of claim17, wherein the fusion polypeptide further comprises an NSI antigen oran immunogenic fragment thereof.
 25. The composition of claim 17,wherein the Mycobacterium species is Mycobacterium tuberculosis.
 26. Amethod for detecting tuberculosis in a patient comprising the steps of:(a) contacting dermal cells of a patient with one or more polypeptidesencoded by a nucleic acid of claim 1, and (b) detecting an immuneresponse on the patient's skin and therefrom detecting tuberculosis inthe patient.
 27. The method of claim 26, wherein the immune response isinduration.
 28. The method of claim 26, wherein the Mycobacteriumspecies is Mycobacterium tuberculosis.
 29. A diagnostic kit comprising:(a) a polypeptide encoded by a nucleic acid of claim 1; and (b)apparatus sufficient to contact the polypeptide encoded by nucleic acidwith the dermal cells of a patient.
 30. A method for eliciting an immuneresponse in a mammal, the method comprising the step of administering tothe mammal an immunologically effective amount of a nucleic acidencoding a MTB32A antigen and a MTB39 antigen from a Mycobacteriumspecies of the tuberculosis complex, wherein said nucleic acidhybridizes under highly stringent conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:1 or a complement thereof,and wherein the MTB32A antigen has a mutation at amino acid position 183as compared to wild type MTB32A.
 31. The method of claim 30, wherein themutation is serine to alanine.
 32. The method of claim 30, wherein themammal has been immunized with BCG.
 33. The method of claim 30, whereinthe mammal is a human.
 34. The method of claim 30, wherein thecomposition is administered prophylactically.
 35. The method of claim30, wherein the nucleic acid comprises nucleotide sequence SEQ ID NO:1.36. The method of claim 30, wherein the nucleic acid encodes an aminoacid sequence of SEQ ID NO:2.
 37. The method of claim 30, wherein thenucleic acid comprises nucleotide sequence SEQ ID NO:3.
 38. The methodof claim 30, wherein the nucleic acid encodes an amino acid sequence ofSEQ ID NO:4.
 39. A method for eliciting an immune response in a mammal,the method comprising the step of administering to the mammal animmunologically effective amount of a composition comprising a MTB32Aantigen and a MTB39 antigen from a Mycobacterium species of thetuberculosis complex encoded by a nucleic acid that hybridizes underhighly stringent conditions to a nucleic acid comprising a nucleotidesequence of SEQ ID NO:1 or a complement thereof, or an immunogenicfragment thereof, wherein the MTB32A antigen has a mutation at aminoacid position 183 as compared to wild type MTB32A.
 40. The method ofclaim 39, wherein the mutation is serine to alanine.
 41. The method ofclaim 39, wherein the nucleic acid comprises nucleotide sequence SEQ IDNO:1.
 42. The method of claim 39, wherein the nucleic acid encodes anamino acid sequence of SEQ ID NO:2.
 43. The method of claim 39, whereinthe nucleic acid comprises nucleotide sequence SEQ ID NO:3.
 44. Themethod of claim 39, wherein the nucleic acid encodes an amino acidsequence of SEQ ID NO:4.
 45. The method of claim 39, wherein the mammalhas been immunized with BCG.
 46. The method of claim 39, wherein themammal is a human
 47. The method of claim 39, wherein the composition isadministered prophylactically.
 48. An isolated nucleic acid encoding afusion polypeptide comprising a MTB32A antigen, a MTB39 antigen, and aMTB85B antigen from a Mycobacterium species of the tuberculosis complexwherein said nucleic acid hybridizes under highly stringent conditionsto a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3 or acomplement thereof, and wherein the MTB32A antigen has a mutation atamino acid position 183 as compared to wild type MTB32A.
 49. The nucleicacid of claim 48, wherein the mutation is serine to alanine.
 50. Thenucleic acid of claim 48, wherein the MTB85B antigen is from M bovis andthe MTB32A antigen and the MTB39 antigen are from M. tuberculosis. 51.The nucleic acid of claim 48, comprising a nucleotide sequence SEQ IDNO:3.
 52. The nucleic acid of claim 48, encoding an amino acid sequenceof SEQ ID NO:4.
 53. An expression vector comprising the nucleic acid ofclaim
 48. 54. A host cell comprising the expression vector of claim 53.55. The host cell of claim 54, wherein the host cell is selected fromthe group consisting of E. coli, yeast, and mammalian cells.
 56. Acomposition comprising a nucleic acid according to claim 48 and aphysiologically acceptable carrier.
 57. The composition of claim 56,wherein the fusion polypeptide encoded by the nucleic acid furthercomprises an NS1 antigen or an immunogenic fragment thereof.
 58. Anisolated fusion protein encoded by the nucleic acid of claim
 48. 59. Acomposition comprising a fusion protein of claim 58, and aphysiologically acceptable carrier.
 60. The composition of claim 59,comprising a non-specific immune response enhancer.
 61. The compositionof claim 60, wherein the non-specific immune response enhancer is anadjuvant.
 62. The composition of claim 61, wherein the adjuvantcomprises QS21 and MPL.
 63. The composition of claim 61, wherein theadjuvant comprises QS21, MPL, and CpG.
 64. The composition of claim 61,wherein the adjuvant is selected from the group consisting of ENHANZYN,MPL, 3D-MPL, IFA, QS21, CWS, TDM, AGP, CPG, Leif, saponin, and saponinmimetics.
 65. The composition of claim 59, further comprising BCG orpVac.
 66. The composition of claim 59, wherein the fusion polypeptidefurther comprises an NS1 antigen or an immunogenic fragment thereof. 67.A method for detecting tuberculosis in a patient, said methodcomprising: (a) contacting dermal cells of a patient with one or morepolypeptides encoded by a nucleic acid of claim 48, and (b) detecting animmune response on the patient's skin and therefrom detectingtuberculosis in the patient.
 68. The method of claim 67, wherein theimmune response is induration.
 69. A diagnostic kit comprising: (a) apolypeptide encoded by a nucleic acid of claim 48; and (b) apparatussufficient to contact the polypeptide encoded by nucleic acid with thedermal cells of a patient.
 70. A method for eliciting an immune responsein a mammal, the method comprising the step of administering to themammal an immunologically effective amount of a nucleic acid encoding aMTB32A antigen, a MTB39 antigen, and a MTB85B antigen from aMycobacterium species of the tuberculosis complex, wherein said nucleicacid hybridizes under highly stringent conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:3 or a complement thereof,wherein the MTB32A antigen has a mutation at amino acid position 183 ascompared to wild type MTB32A.
 71. The method of claim 70, wherein themutation is serine to alanine.
 72. The method of claim 70, wherein themammal has been immunized with BCG.
 73. The method of claim 70, whereinthe mammal is a human.
 74. The method of claim 70, wherein thecomposition is administered prophylactically.
 75. The method of claim70, wherein the nucleic acid comprises nucleotide sequence SEQ ID NO:3.76. The method of claim 70, wherein the nucleic acid encodes an aminoacid sequence of SEQ ID NO:4.
 77. A method for eliciting an immuneresponse in a mammal, the method comprising the step of administering tothe mammal an immunologically effective amount of a compositioncomprising a MTB32A antigen, a MTB39 antigen, and a MTB85B antigen froma Mycobacterium species of the tuberculosis complex encoded by a nucleicacid that hybridizes under highly stringent conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:3 or a complement thereof,or an immunogenic fragment thereof, wherein the MTB32A antigen has amutation at amino acid position 183 as compared to wild type MTB32A. 78.The method of claim 77, wherein the mutation is serine to alanine. 79.The method of claim 77, wherein the nucleic acid comprises nucleotidesequence SEQ ID NO:3.
 80. The method of claim 77, wherein the nucleicacid encodes an amino acid sequence of SEQ ID NO:4.
 81. The method ofclaim 77, wherein the mammal has been immunized with BCG.
 82. The methodof claim 77, wherein the mammal is a human.
 83. The method of claim 77,wherein the composition is administered prophylactically
 84. An isolatednucleic acid encoding a fusion protein, the nucleic acid comprising anucleotide sequence selected from the group consisting of MTB89F (SEQ IDNO:6), MTB83F (SEQ ID NO:7), MTB81F (SEQ ID NO:8), MTB114F (SEQ IDNO:9), MTB102tm2F (SEQ ID NO:10) and MTB103F (SEQ ID NO:11).
 85. Afusion protein comprising an amino acid sequence selected from the groupconsisting of MTB89F (SEQ ID NO:13), MTB83F (SEQ ID NO:14), MTB81F (SEQID NO: 15), MTB 114F (SEQ ID NO:16), MTB102tm2F (SEQ ID NO:17) andMTB103F (SEQ ID NO:18).